Voltage dependence of tonic GABA conductance
The voltage-dependence of tonic inhibition was evaluated in cultured hippocampal neurons by measuring tonic current amplitude over a range of membrane potentials from −100 mV to +40 mV. Attention was focused on larger neurons (whole-cell capacitance range = 43–100 pF) and recording conditions were designed to isolate GABAA
receptor currents. Representative current traces recorded at −40 mV and +40 mV during bicuculline application are presented in . Tonic current amplitude was defined as the change in mean holding current induced by application of 10 µM bicuculline. Mean holding current was determined from Gaussian fits to all-points amplitude histograms constructed from segments of current data (). Bicuculline was applied within 30 seconds of changing membrane potential. As shown previously, there was a significant amount of tonic current in cultured neurons (Wu et al., 2001
). Because neurons were continuously superfused by bath solution, this presumably resulted from activation of GABAA
receptors by ambient GABA that was trapped between the recorded neurons and other neurons, glia, or the culture dish. Tonic currents were outwardly-rectifying, seen by comparing currents measured at +40 mV and −40 mV (; EGABA
~ 0 mV) and in the I–V curve for tonic current (). Tonic current amplitudes in are the mean of 1–3 measurements at each potential. Calculation of chord conductance (using the theoretical ECl
value of 0 mV as EGABA
) showed that tonic GABA conductance increased in a near-linear manner with membrane depolarization (). Summary data for current and conductance (normalized to membrane capacitance) also showed outward rectification and a near-linear increase in conductance (; n=12 cells). The mean specific conductance was 15±3 pS/pF at −80 mV and 29±5 pS/pF at −40 mV (p<0.01). On average, tonic GABA conductance of hippocampal neurons increased with depolarization by 0.32 pS*pF−1
The prolonged membrane depolarization used above to measure tonic current could potentially produce changes in intracellular Cl− and HCO3− concentrations. This could increase current amplitude through changes in electrochemical driving force rather than an actual increase in conductance. To evaluate this possibility, cells were held at −60, −20, and +40 mV for 2–3 minutes and then given voltage ramps (−60 mV to +40 mV over 2.5 s) before and during bicuculline exposure. Point-by-point subtraction of these records yielded the “bicuculline-sensitive” ramp current and allowed determination of tonic current reversal potential (i.e. EGABA) (data not shown). The reversal potential for tonic current was not significantly different when measured from holding potentials of −60 mV, −20 mV, or +40 mV (−0.1±0.8 mV, 0.1±1.0 mV, and 2.0±0.9 mV, respectively; n=8, p-values of 0.81 and 0.13). Thus, changes in EGABA cannot account for the increased tonic current amplitudes seen with depolarization. For changes in driving force alone to account for the increased tonic current amplitude seen with depolarization from −40 mV to +40 mV in the neuron in , EGABA would have needed to shift by +24 mV (much larger than the insignificant shift of +2 mV seen experimentally).
Similar results were obtained using SR95531 (SR, 100 µM) as the GABAA receptor antagonist to measure tonic current. Tonic currents measured with SR were outwardly-rectifying and the capacitance-specific conductance increased during depolarization with a slope of 0.21 pS*pF−1*mV−1 (n=4). The mean capacitance-specific tonic conductance seen with SR was 8±2 pS/pF and 15±4 pS/pF at −80 mV and −40 mV, respectively. Although the capacitance-specific conductance seen in experiments with SR was lower than values obtained with bicuculline, with either antagonist there was a ≈2-fold increase in tonic conductance when cells were depolarized from −80 to −40 mV. Because these data with SR were obtained from a different subset of neurons than those with bicuculline, it is not possible to directly compare the absolute conductance between the two datasets. The lower conductance seen with SR may be due to variability between hippocampal cultures.
Effect of reducing vesicular and non-vesicular GABA release on the voltage-dependence of tonic currents
The increase in tonic conductance seen with depolarization could be due to elevated ambient [GABA]. Ambient GABA may increase during membrane depolarization through reduction of driving force for GABA uptake into the recorded neuron or nonvesicular release of GABA by reversal of GABA transport (Richerson and Wu, 2003
; Wu et al., 2007
). Because there was no GABA in our intracellular recording solution, nonvesicular release from the depolarized neuron was not expected to occur, but it is possible that depolarization would reduce GABA uptake. To assess the contribution of GABA transporters to the voltage-dependence of tonic inhibition, we measured tonic current in the presence of the GAT1 inhibitor SKF-89976A (SKF). SKF inhibits GABA transport in these neurons with an IC50
of 1.3 µM and >80% inhibition is produced by 10 µM (Wu et al., 2007
). To ensure maximal block of GABA transport we used a high concentration of SKF (40 µM). The current response to exogenous GABA (0.5 µM) was not affected by 40 µM SKF (104±8% of control, n=7, p=0.65; data not shown), indicating that SKF did not act directly on GABAA
receptors. As previously reported (Rossi et al., 2003
; Wu et al., 2003
), application of SKF induced an inward current that reached a peak within 15–20 seconds (average peak amplitude of −283±68 pA, n=6) and decayed to a steady-state within 60 seconds. This inward current was completely blocked by bicuculline (10 µM) indicating that it was due to elevated ambient GABA acting on GABAA
receptors (). At −60 mV, tonic current density was reversibly increased by SKF from −0.91±0.11 pA/pF to −2.05±0.15 pA/pF (n=6, p-value <0.01). Mean current density and capacitance-specific conductance from cells studied with SKF are plotted against voltage in . In the presence of SKF, tonic currents displayed outward rectification and the near-linear relationship between tonic conductance and membrane potential seen under control conditions persisted. The specific conductance in the presence of SKF increased from 29±3 pS/pF to 46±9 pS/pF when neurons were depolarized from −80 mV to −40 mV. The solid lines in are linear fits to the data with slopes of 0.31 pS*pF−1
and 0.33 pS*pF−1
for control and SKF, respectively. Thus, there was a shift in conductance to higher levels due to increased ambient [GABA], without a change in the voltage dependence. The GAT-3 inhibitor SNAP-5114 (100 µM) alone or in combination with SKF did not affect the voltage dependence of tonic current (n=3, data not shown).
Vesicular release of GABA also regulates tonic current amplitude (Glykys and Mody, 2007
). Depolarization of a recorded neuron could increase vesicular release from the same neuron or from neighboring cells. The inclusion of QX-314 in our pipette solution would limit action potential mediated vesicular release of GABA from recorded neurons. Furthermore, recordings were made from large, pyramidal-appearing neurons so most neurons studied were probably not GABAergic. In hippocampal neurons, evoked IPSCs between neuron pairs are completely blocked by zero extracellular Ca2+
(Wu et al., 2006
). The frequency and amplitude of miniature IPSCs are also greatly reduced in the absence of extracellular Ca2+
to 25% and 18% of control, respectively (Wu et al., 2006
), and the residual vesicular release is not increased by depolarization. To ensure that tonic conductance was not increased by depolarization as a result of evoked vesicular GABA release, tonic current was measured in bath solution containing 1 mM EGTA and no Ca2+
(). In contrast to the reduction of phasic currents, tonic current amplitude was unchanged in zero Ca2+
/1 mM EGTA () nor was the outward rectification and voltage-dependent increase of tonic conductance, implying that these phenomena were not caused by depolarization-evoked vesicular release of GABA. These experiments also excluded a contribution of Ca2+
entry via voltage-gated calcium channels to the voltage-dependence of tonic currents.
Voltage-dependence of current evoked with exogenous GABA
Inhibition of GABA transport or vesicular GABA release did not affect the increase in tonic current with depolarization, suggesting that this was not due to an elevation of ambient [GABA]. To assess the voltage-dependence of GABAA
receptors, exogenous GABA (1 µM) was applied to neurons held at a range of membrane potentials (−100 mV to +40 mV). Current amplitude was measured early during GABA application (near peak current at −60 mV) and near steady state. As previously reported for hippocampal neurons (Segal and Barker, 1984
; Ashwood et al., 1987
; Yoon, 1994
; Pytel et al., 2006
), GABA-evoked currents were outwardly-rectifying (). The outward rectification was most pronounced when currents were measured at the end of a 10 s GABA application (). A GABA concentration of 1 µM is not selective for extrasynaptic/perisynaptic receptors that mediate tonic currents, but the steady-state currents induced by this level of GABA are most relevant to tonic activation of these receptors. These data also show an acceleration of desensitization with hyperpolarization and slow, continuous activation of current at +40 mV () (Yoon, 1994
). This latter feature was seen with GABA concentrations of 1–30 µM.
Figure 3 Currents evoked with exogenous GABA were outwardly-rectifying. A: Current traces during GABA application (1 µM) at different holding potentials (as indicated). Baselines are adjusted to allow comparison of currents. Note the acceleration of desensitization (more ...)
To compare the voltage-dependence of endogenous tonic currents and currents evoked with exogenous GABA we calculated the ratio of current at +40 mV to current at −40 mV (i.e. rectification ratio; ). The rectification ratio of currents evoked with exogenous GABA (1 µM) ranged between 1.93±0.2 for currents measured early during GABA application (near peak current at −60 mV) to 3.35±0.5 for currents measured near steady-state (n=5). These values were not significantly different from endogenous tonic currents under control conditions (rectification ratio 2.56±0.3, range=1.63–4.37, n=12). No significant changes in rectification ratio of tonic currents were seen in the presence of SKF (1.89±0.1, range=1.58–2.48, n=6; p=0.11) or with zero Ca2+/1 mM EGTA (2.78±0.5, range=1.70–4.73, n=6; p=0.46) compared to control conditions. The similar outward rectification of endogenous tonic currents and currents evoked with exogenous GABA is consistent with the voltage-dependence of tonic currents being primarily due to intrinsic properties of extrasynaptic GABAA receptors rather than to elevation of ambient GABA.
Post-depolarization potentiation (PDP) of GABA currents
While examining the voltage-dependence of GABA current, it was found that after GABA was applied at +40 mV, subsequent GABA currents at −60 mV were increased. To characterize this effect, repeated applications of GABA (1 µM) were made every 30 s at a holding potential of −60 mV. After a stable baseline was established, GABA was applied while neurons were depolarized to +40 mV (total depolarization time was 15 s). Neurons were then repolarized to −60 mV and the GABA response was measured again (). On average, the peak and steady-state current amplitudes measured 30 s after repolarization were increased by 55±7% and 52±9%, respectively (n=5). This post-depolarization potentiation (PDP) of GABA current persisted for up to 120 s in some cells (see ). Recovery from PDP followed an exponential time course. The time constant for recovery of PDP was estimated by normalizing current at −60 mV to baseline values and fitting a single exponential function to these data (). The time constant for recovery of PDP was 25 s for the experiment illustrated () and was the same for summary data from 5 cells (). PDP was concentration-dependent. With 3 µM GABA, steady-state currents measured 30 s after repolarization were potentiated by 131±9% (n=4) compared to 52±9% with 1 µM GABA (n=5; p<0.05). PDP was also seen with low intracellular Cl−
(15 mM) and HCO3
-free (HEPES buffer) bath solution (Supplemental Figure 1
). Experiments with low intracellular Cl−
were performed with 3 µM GABA. PDP with low intracellular Cl−
averaged 161±35% (n=5) which was not different from PDP seen with 3 µM GABA and 135 mM intracellular Cl−
(131±9%, n=4, p=0.48) (Supplemental Figure 1
Figure 4 Post-depolarization potentiation (PDP) of GABA currents. A: Current recordings during repeated GABA applications (1 µM, 5 s) at indicated times (holding potential −60 mV). At t=0 GABA was applied at +40 mV (note the slow activation of (more ...)
Time- and voltage-dependence of PDP
To determine the minimum duration of depolarization required to potentiate GABA currents, neurons were depolarized to +40 mV for 1–4.5 s during GABA application (). Current was measured 1.5 s after repolarization to −60 mV and normalized to current at the corresponding time point during GABA application without depolarization. With 1 µM GABA the mean potentiation was 11±9% and 82±19% following depolarization of 1 s and 4.5 s, respectively (n= 4; ). For the cell shown in , current after a 2.5 s depolarization was larger than the peak current produced by GABA application alone, suggesting that PDP was not simply reversal of desensitization.
The voltage-dependence of PDP was examined by depolarizing neurons from a holding potential of −60 mV to voltages between −30 mV and +30 mV (10 mV steps, 6.5 s duration) during GABA application (3 µM; ). Current was measured 1.5 s after repolarization to −60 mV and normalized to control current. Current potentiation was 13±3% (n=4) after depolarization to −30 mV even though the current continued to desensitize, suggesting that potentiation represents a different process than reversal of desensitization. Current potentiation increased progressively with greater depolarization reaching 99±19% at +30 mV (). PDP was seen after depolarization to voltages at which little net current flow occurred (i.e. −10 mV to +10 mV), implying that PDP of GABA current was independent of intracellular anion accumulation during depolarization (see below).
PDP of tonic currents
PDP of tonic currents was investigated with continuous application of a low concentration of exogenous GABA (0.3 µM) to provide more selective activation of high-affinity extrasynaptic/perisynaptic GABAA receptors. The use of exogenous GABA would also minimize any contribution of voltage-dependent alterations in ambient [GABA] to PDP. Application of 0.3 µM GABA increased tonic current by −314±42 pA (n=11). Tonic current was measured at −60 mV with repeated application of bicuculline (10 µM, ~5 s) (). After establishing a baseline, neurons were depolarized to +40 mV for 30 s, and tonic current was again measured after repolarization (). Tonic current at −60 mV was increased by transient depolarization in all cells tested (n=11). Over the duration of a typical experiment (15–20 minutes), there was a progressive reduction in tonic current amplitude to −159±18 pA, apparently due to rundown and/or desensitization of extrasynaptic GABAA receptors (). To account for this rundown/desensitization, tonic currents were normalized to the value preceding each depolarization. Peak potentiation under control conditions (measured 3–8 s after repolarization) averaged 122±13% (range 57–240%, n=11). Current rundown/desensitization was not associated with a reduction in PDP (p=0.44 for comparison of 1st and 2nd control depolarizations, n=11). PDP of tonic current recovered with a time course well described by a single exponential function (). The time constant for recovery averaged 45±4 s (range 23–78 s, n=11). PDP of tonic currents also occurred with smaller depolarizations to −20 mV (37±12%, n=4, data not shown).
Figure 6 PDP of tonic currents. A: Tonic currents in the presence of low levels of exogenous GABA (0.3 µM). Upward deflections (e.g. the one indicated by solid arrow) represent response to 10 µM bicuculline (Bic) used to measure tonic current amplitude. (more ...)
PDP was attenuated when neurons were depolarized in the presence of the GABAA receptor antagonists SR or bicuculline (). Wash of these antagonists began 1–3 s before repolarization and care was taken to allow complete wash of the antagonists prior to measuring tonic current (for example, see the right panel in ). Wash of SR was slower than bicuculline but was still complete within 10 s. Because of this, PDP was measured at variable times after depolarization with antagonists. To compare PDP under control conditions and after depolarization in the presence of antagonists, normalized tonic currents were aligned so that repolarization occurred at t=0 (). With this method, it can be seen that SR application during depolarization reduced PDP for more than 60 s after repolarization (), much longer than the time required for wash of SR. Mean potentiation is plotted as a function of time after repolarization in . The first measurement of PDP after depolarization in the presence of SR was significantly reduced compared to both the first and second measurements under control conditions (n=5, p<0.01) (). With bicuculline, PDP was also significantly reduced at all three time points compared to the corresponding control measurements (p<0.01, n=6). If incomplete wash of bicuculline after the prolonged application was the cause of PDP antagonism then the percent inhibition of PDP by bicuculline is predicted to decrease with time (as drug washed off). The percent inhibition produced by bicuculline for the 1st, 2nd, and 3rd measurements of tonic current after repolarization was 62±12%, 66±12%, and 83±16% (n=6). The trend for greater inhibition at the 3rd measurement was not significant (p=0.11). Thus, the reduction in PDP during application of antagonists was not due to incomplete washout of the drugs. The time constants for recovery of PDP following depolarization in the presence of SR (66±17 s, n=5) or bicuculline (47±8 s, n=6) were not significantly different from control (45±4 s, n=11, p=0.34 and 0.89, respectively).
Tonic current due to endogenous GABA was also persistently potentiated by depolarization (). In the absence of any exogenous GABA, tonic current was increased by >10% in response to transient depolarization in 67% (8/12) of cells tested, compared to 100% (11/11) of cells tested with 0.3 µM exogenous GABA. In cells that displayed PDP with endogenous GABA (defined as a >10% increase in current), the peak potentiation (75±17%, range 32–165, n=8) was smaller than seen with 0.3 µM GABA (121±13%, n=11, p=0.04). PDP with endogenous GABA recovered with a mean time constant of 63±15 s (; range 25–135 s, n=8) and was antagonized by SR (100 µM) exposure during depolarization (). Transient application of exogenous GABA (1 µM) for 5 s during neuronal depolarization only enhanced the subsequent PDP of endogenous tonic currents that occurred upon repolarization (). PDP of endogenous tonic current was also seen in the presence of SKF (40 µM) (153±53% potentiation, n=5) and in solutions with zero Ca2+/1 mM EGTA (52±2% potentiation, n=4) (data not shown).
Figure 7 PDP of tonic currents due to endogenous GABA. A: Tonic currents due to endogenous GABA before and after depolarization to +40 mV. Period of depolarization is clipped to display the PDP of tonic current. Upward deflections (e.g. the one indicated by arrow) (more ...)
PDP was due to increased GABA conductance
The outward current produced by GABAA receptor activation at +40 mV represents an inward movement of anions and could lead to intracellular anion accumulation. The contribution of intracellular anion accumulation (and changes in electrochemical driving force) to PDP was evaluated by recording membrane currents in response to voltage ramps in the presence and absence of exogenous GABA (1–3 µM) (). Difference currents were obtained by subtracting these records and used to determine the reversal potential of GABA-evoked currents (i.e. EGABA). To reproduce the timing previously used to measure PDP (), difference currents were determined at baseline (t =−30 s) and then 30 s after GABA application at +40 mV (t=30 s) (). Chord conductance at −60 mV was calculated using the measured value of EGABA. We also determined slope conductance from ramp currents using a linear fit to data between −60 mV and −10 mV. For the experiment illustrated in GABA was −3.0 mV prior to depolarization. Thirty seconds after GABA was applied at +40 mV, EGABA was shifted to +3.6 mV indicating intracellular anion accumulation. The chord conductance was 17.2 nS under baseline conditions and 32.2 nS after depolarization, an 87% increase. Similar values were obtained from the slope conductance (a parameter that is independent of EGABA, values indicated in ). On average, EGABA shifted from −4.9±0.9 mV at baseline to +2.9±0.9 mV post-depolarization (n=5). Thus, the increased current during PDP cannot be accounted for simply by changes in driving force due to anion accumulation.
Figure 8 PDP of GABA current was due to increased conductance. A: Membrane currents in response to voltage ramps before (control) and after GABA application (1 µM). The GABA-evoked ramp current (“difference current”) was obtained from point-by-point (more ...)
PDP of conductance was also seen with physiological intracellular Cl− concentration ([Cl−]in=15 mM; ). With this lower [Cl−]in, EGABA was −59.8±2 mV at baseline and shifted to −28.5±5 mV 30 s after GABA (3 µM) was applied to depolarized neurons (n=5). Slope conductance of the neuron in increased from 13 nS at baseline to 33 nS at t=30. Both slope and chord conductances were significantly increased after GABAA receptor activation at +40 mV with high or low intracellular Cl− concentrations (p<0.05, n=5 cells at each Cl− concentration) (). In experiments with low [Cl−]in, chord conductance was calculated at −70 mV and slope conductance was determined with linear fits to data from −70 to −20 mV. PDP of GABA conductance was also seen in HCO3−-free bath solution (HEPES). The average chord conductance at −70 mV increased from 16.6 nS at baseline to 33.1 nS after depolarization in HEPES (n=2). There was no significant difference between the potentiation of slope conductance with low [Cl−]in (mean 86±26%, range 23–154%, n=5) and high [Cl−]in (mean 51±13%, range 20–94%, n=5, p-value=0.34).
Because PDP of GABA current was observed within seconds of repolarization (see ), it is possible that anion accumulation at these time points could be larger than that measured 30 s after repolarization. To evaluate this, chord conductance was measured before and after step depolarization in the presence of GABA (Supplemental Fig. 2
). These data indicate that 1 s after repolarization, when anion accumulation should be maximal, PDP was associated with absolute increases in conductance.
Time course for PDP of tonic conductance
To determine the time course of tonic conductance changes produced by depolarization, we simultaneously measured reversal potential and tonic current amplitude in the presence of 0.3 µM exogenous GABA. A voltage ramp protocol (−30 to +30 mV over 500 ms) was repeated every 10 s from a holding potential of −60 mV. Bicuculline was applied at 30–50 s intervals and traces recorded in the presence of bicuculline were averaged and subtracted from control sweeps to yield tonic current amplitudes at −60 mV and bicuculline-sensitive ramp currents. illustrates bicuculline-sensitive ramp currents recorded during a baseline period at −60 mV, during depolarization to +40 mV, and then following repolarization to −60 mV (times of ramp currents indicated in figure). During depolarization, there was a progressive shift of reversal potential (i.e. EGABA) towards positive voltages that stabilized near +6 mV after 110 s of depolarization to +40 mV (experiment time of 260 s) (). Upon return to −60 mV, EGABA recovered towards control values over several minutes. Tonic conductance increased in parallel to EGABA during depolarization to +40 mV and continued to increase after EGABA approached a plateau (). Tonic conductance of the cell illustrated in peaked at 19.1 nS during depolarization and underwent a rapid drop to 8.7 nS upon return to −60 mV, but remained above the baseline value of 3.2 nS. PDP of tonic conductance following this prolonged (150 s) depolarization recovered to baseline with a time constant of 104 s (). Similar results were obtained from other cells studied in this manner (n=4).
Figure 9 Time course for PDP of tonic conductance. A: Bicuculline-sensitive ramp currents. Ramp currents were recorded every 10 s, first at a holding potential of −60 mV, during depolarization to +40 mV, and then after repolarization to −60 mV. (more ...)
To assess the contribution of anion accumulation to PDP, we calculated the theoretical changes in tonic current that would result solely from shifts in EGABA for comparison to the observed changes in tonic current. Without any change in conductance, tonic current would decrease during depolarization as intracellular Cl−/HCO3− accumulated (, solid line). This is in contrast to the slow increase in tonic current seen experimentally (, open circles). Immediately following repolarization, tonic current would be increased by only 45 pA if there was only a shift of EGABA without conductance changes, a small increase compared to the observed increase in tonic current of 425 pA. Conversely, using experimentally determined chord conductance values we calculated the theoretical tonic current that would have resulted if EGABA remained constant (, solid triangles). Given a chord conductance of 8.7 nS at the peak of PDP (Itonic = −590 pA, EGABA = +7.6 mV), the peak tonic current would be −463 pA if EGABA had remained constant at −7 mV. These calculations indicate that an increase in conductance was the dominant reason for the increase in current, and changes in electrochemical driving force due to anion accumulation contributed no more than 10–30% to PDP of tonic current.
PDP was not due to direct effects of elevated intracellular Cl− on GABAA receptors
The results presented above demonstrate an absolute increase in whole-cell conductance during PDP of GABA current, independent of driving force changes due to anion accumulation (Chabwine et al., 2004
). In addition to effects on driving force, elevations of [Cl−
] can directly increase the unitary conductance of GABAA
receptors (Bormann et al., 1987
; Fatima-Shad and Barry, 1993
). The similarity between the time course for recovery of EGABA
and conductance during PDP seen in raised the possibility that modulation of single-channel conductance by Cl−
ions contributed to PDP. To evaluate this possibility, we measured GABA currents (1 µM) at −60 mV every 30 s before and after GABA was applied during depolarization. In these experiments, however, GABA was co-applied to depolarized neurons with a low Cl−
bath solution (Na-methanesulfonate substitution for NaCl, 3 mM Cl−
) so that GABA would produce inward currents (outward movements of anions) even at +40 mV (). A transient outward current was seen at +40 mV as the low Cl−
solution washed off while the receptors were still deactivating. The net charge movement induced by GABA in low Cl−
solutions was determined by integrating baseline-adjusted traces recorded at +40 mV. The net charge movement produced by this procedure averaged −2.0±0.5 nC (n=7), indicating a net outward movement of anions despite the transient outward current seen with solution exchange during receptor deactivation (). PDP still occurred after GABA receptor activation in low Cl−
solution (39±6%, n=7), indicating that anion accumulation is unnecessary for the production of PDP (). The amount of PDP seen with low Cl−
solutions was not significantly different from that in cells on the same coverslips using normal bath solution (42±4%, n=3, p=0.80). Transient depolarization alone (without GABA application) also potentiated GABA currents at −60 mV (16±6%, n=3) but this was reduced compared to that seen with GABA application during depolarization with normal or low Cl−
bath solution in cells from the same coverslips (p=0.02 and 0.05, respectively) (). Depolarization alone did not affect EGABA
(see above). These data support the conclusion that PDP resulted primarily from direct effects of membrane potential on GABAA
receptor function rather than the effects of anion accumulation on driving force and single channel conductance. Macroscopic GABA currents in outside-out patches also underwent PDP, adding further support to the conclusion that PDP resulted from intrinsic receptor properties independent of anion redistribution or cellular factors (Supplemental Figure 3
Potentiation of extrasynaptic GABAA receptors during epileptiform activity
To determine whether a physiological or pathophysiological pattern of depolarization can cause PDP of GABAA receptors, we recorded spontaneous epileptiform bursts during current-clamp experiments in the presence of 4-aminopyridine (100 µM) (). We then used this waveform as a voltage clamp command. This waveform had a maximum action potential frequency of 40 Hz and mean membrane potential of −25 mV. When this stimulus was used during application of exogenous GABA (1–10 µM), current was potentiated following repolarization (). Current was measured 0.2 s and 2 s following the depolarizing stimulus and compared to control current (with no depolarization). Two hundred milliseconds after the depolarizing stimulus, currents were increased by 40.5±9% and 125±25% with 1 µM and 10 µM GABA, respectively (, n=4 cells at each concentration, p<0.01). Tonic currents induced by 0.3 µM exogenous GABA were also potentiated (defined as >10% increase) by the pathophysiologic depolarizing stimulus in 7/10 cells (). Tonic currents were increased by 21±3% and 5±4% when measured 2 s and 20 s after the depolarizing stimulus, respectively (, n=7). Similar to results with step depolarizations, the frequency of PDP with epileptiform depolarization was greater in the presence of exogenous GABA. With endogenous GABA, PDP of tonic currents was seen in only 3/10 neurons following epileptiform depolarization, consistent with the smaller magnitude of PDP seen with lower GABA concentrations. While the potentiation seen with epileptiform bursts was smaller than that produced with step depolarization, these results demonstrate that persistent, voltage-dependent changes in the function of GABAA receptors underlying tonic conductance are likely to occur during pathophysiological neuronal discharges.
Figure 11 GABA current was potentiated by an epileptiform burst. A: Membrane potential of a hippocampal neuron in the presence of 4-aminopyridine (100 µM). Bursts of high frequency action potentials occur spontaneously. Data enclosed in the dashed box was (more ...)